Proper cell cycle transitions are driven by coordinated waves of ubiquitin-dependent degradation of key cell cycle regulators by APC/C and SCF E3 ubiquitin ligase complexes. Among them, SCF?-TRCP is one of the well-characterized Cullin 1-based E3 ubiquitin ligases involved in numerous important cellular processes through promoting the degradation of critical regulatory proteins including ?-catenin, Emi1 and I?Bs. During the last funding cycle, our group and others have made significant contributions to further our understanding of the critical role of ?-TRCP in various physiological functions such as autophagy, cell migration, DNA damage response and cell cycle regulation by defining DEPTOR, VEGFR, Mdm2 and Cdh1, respectively, as downstream substrates of SCF?-TRCP. However, it remains largely unknown how upstream signaling pathways control ?-TRCP stability and physiological functions in vivo. To this end, our preliminary results reveal that like Fbw7, ?-TRCP undergoes auto-ubiquitination to negatively control its own stability, while OTUD3, but not other OTU family of DUBs, specifically interacts with, and deubiquitinates, ?-TRCP to control its stability. As such, depletion of OTUD3 significantly reduced ?-TRCP abundance. In Aim #1, we intend to explore mechanistically how the ?-TRCP signaling pathway is governed by the dynamic auto-uibiquitination and deubiquitination processes to influence biological functions of ?-TRCP in vivo. Furthermore, other than tissue context-dependent roles for ?-TRCP in tumorigenesis, the physiological role of ?-TRCP in metabolism such as lipid homeostasis has not been described. We reasoned that identification of additional ?-TRCP ubiquitin substrate(s) would further define its physiological functions. To overcome the concern of using ectopic overexpression conditions in most E3 ligase-substrate screenings, we developed a novel screening system to identify ?-TRCP substrates at endogenous levels using a ?-TRCP phospho-degron specific antibody- mediated mass spectrometry approach. We identified many known ?-TRCP targets, validating this screening method, and characterized Lipin1 and Lyric as novel ?-TRCP substrates. This finding provides a novel link between ?-TRCP and tissue-specific metabolic phenotypes observed in ?-TRCP1-/- mice. Therefore, another major focus is to explore mechanistically how ?-TRCP controls hepatocyte lipid metabolism through regulating Lipin1 protein stability and subsequent inhibition of SREBP1 transcriptional activity (Aim # 2). Lastly, we also intend to reveal a critical physiological role for ?-TRCP in controlling enterocyte lipid absorption pathways and tumorigenesis by governing Lyric protein stability (Aim #3). We believe that these proposed studies will significantly extend our understanding of how ?-TRCP exerts tissue context-dependent roles to control important process such as lipid metabolic pathways, and further implicate that in addition to tumorigenesis, aberrant regulation of ?-TRCP signaling pathway may lead to other human diseases including lipid homeostasis disorders, which will ultimately provide the rationale to develop better therapies.